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Abstract

Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H+] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.

Pick a few single colonies from
the agar plate and use to inoculate 2 x 250 ml conical flasks, each
containing 100 ml of LBK liquid medium supplemented with 100 μg/ml
carbenicillin. Grow the cultures overnight (~15 h) at 30 °C with 250 rpm
shaking in a temperature-controlled shaking incubator. Measure the
optical density at 600 nm (OD600) of the culture using a
spectrophotometer; it should be between 3.0-3.3. If the OD600 is less
than 3.0, then incubate for a further 0.5-1.0 h and re-measure. If the
OD600 is greater than 3.3, the cultures have overgrown and fresh
cultures will need to be prepared.

Inoculate 2 x 5 L conical
flasks, each containing 1,000 ml of LBK liquid medium supplemented with
100 μg/ml carbenicillin, with 15 ml of overnight culture. Incubate at 32
°C with 270 rpm shaking for ~2.5 h in a temperature-controlled shaking
incubator. The OD600 should be ~0.6. Drop the temperature to 25 °C and
grow with 270 rpm shaking until the OD600 is 1.0. This usually takes
between 0.5 to 1.0 h.

Induce overexpression of MdtM by addition of
0.1% w/v L-(+)-arabinose [5 ml of 20% w/v L-(+)-arabinose] to each
flask. After addition of arabinose, grow the cells for a further 1.5 h
at 25 °C with 270 rpm shaking prior to harvesting.

Transfer the E.
coli TO114 cells that contain overexpressed transporter into 500 ml or
1,000 ml capacity centrifuge pots that have been pre-chilled on ice for
at least 15 min. Pre-cool the centrifuge to 4 °C and harvest cells by
centrifugation at 5,000 x g for 20 min.

Decant the supernatant
and wash the pelleted cells by resuspending in chilled (4 °C) TCDS
buffer. Use 30 ml of TCDS buffer for each one litre of cell culture that
was pelleted. Maintain the cells on ice during this procedure. Harvest
the washed cells by centrifugation as described in step A1 (above) and
repeat the washing procedure. Resuspend the resultant cell pellet in 30
ml of chilled TCDS buffer containing 2 mM PMSF (which should be made up
as a 100 mM stock in ethanol and stored at -20 °C; ensure the solution
is thawed thoroughly and vortexed vigorously before use) and 5 µM DNase
and maintain the mixture on ice.Notes:

Typically, a 1 L culture of bacterial cells will provide sufficient material for these experiments.

Cells should be resuspended either by gentle vortexing using a benchtop
vortexer or by gentle aspiration using a 25 ml sterile plastic pipette.

Decant the resuspended cells into a 100 ml beaker containing a stir
bar, place on magnetic stirrer and stir in a cold room at 4 °C for 20
min. Alternatively, place the beaker on ice in an ice bucket, place on
the magnetic stirrer and stir for 20 min.

The method for
production of inverted vesicles relies on a combination of the fluid
shear forces and decompression created as the cell mixture passes
through the needle valve of a French pressure cell. Generate inverted
membrane vesicles by a single passage of the resuspended cell mixture
through a French pressure cell at a minimum of 4,000 psi. If the
pressure is too low, inverted vesicles will not be formed. The pressure
cell should be chilled on ice for ~30 min prior to use. The resulting
inverted vesicle mixture should be collected in a 100 ml conical flask
kept on ice.

Decant the mixture into a pre-chilled ~50 ml
centrifuge tube and remove any unbroken cells and cell debris by
centrifugation at 18,000 x g for 10 min at 4 °C. Carefully decant the
supernatant (do not disturb the pellet that contains unbroken cells and
cell debris) containing the cell membrane vesicles into a pre-chilled
30-50 ml volume polycarbonate ultracentrifuge tube on ice.

Harvest the inverted vesicles by ultracentrifugation at 100,000 x g for 1
h at 4 °C. Carefully decant the supernatant and retain the pellet.
Place the ultracentrifuge tube containing the pelleted vesicles on ice.

Resuspend the inverted vesicle pellet in 1 ml of ice-cold TCDS buffer
by gentle aspiration using a 1,000 µl pipette. Transfer the resuspended
vesicles to a pre-chilled 1.5 ml Eppendorf tube on ice for use in the
transport assay. In our experience, vesicles stored on ice are stable
for several hours.

Quantify the total membrane protein content of
the inverted vesicles by UV absorbance spectroscopy at 280 nm. Blank
the spectrophotometer using a 10 mm pathlength quartz cuvette containing
1,000 µl of TCDS buffer. The buffer should be at room temperature to
prevent frosting of the cuvette faces. Clean the faces of the cuvette
using a fresh paper wipe prior to measurement. Once the
spectrophotometer is blanked, remove 5 µl of buffer from the cuvette
using a 10 µl pipette and replace with 5 µl of vesicles. Cover the
opening of the cuvette with a square of Parafilm and invert the cuvette a
few times to ensure a homogeneous distribution of vesicles. Record the
absorbance of the vesicle mixture at 280 nm and calculate the total
membrane protein concentration assuming that an A280 of 1.0 is
equivalent to a protein concentration of 1.0 mg/ml.Notes:

Remember to multiply the 280 nm absorbance value you obtain by a factor
of 200 to calculate the concentration of the undiluted vesicle mixture
in mg/ml.

At this stage of the preparation, the resuspended
vesicles can be transferred to tubes in aliquots of 25-100 µl,
snap-frozen in liquid nitrogen and stored at -80 °C for subsequent use.
Vesicles frozen in this way will retain their integrity for several
months. However, if frozen vesicle stocks are used for the subsequent
transport measurements, the vesicles must be thawed very slowly on ice
prior to use to prevent their fracture.

Fluorometric antiport assays

In this section we describe the set-up parameters for a Fluoromax-4
fluorometer. These parameters, however, can form the basis for the
set-up of other fluorometers. Once the instrument is switched on and the
software booted up, set the temperature of the cuvette holder to 25 °C.
Open the instrument software and select for time-based data acquisition
with excitation and emission wavelengths of 492 nm and 525 nm,
respectively. Set the excitation and emission slit widths to 1.5 nm and
2.5 nm, respectively.

Add an aliquot of inverted vesicles (which
should be maintained on ice in TCDS buffer) to room temperature
transport assay buffer containing the acridine orange probe in a 10 mm x
4 mm quartz cuvette to a final concentration of 0.5 mg/ml membrane
protein in a total volume of 1,500 µl. The longest pathlength of the
cuvette should face the excitation light source. Place a small magnetic
flea into the cuvette and stir the contents gently. Allow the vesicles
and assay buffer to equilibrate for ~200 sec.

Start recording the
fluorescence emission. After approximately 50 sec, add 15 µl of 200 mM
stock sodium DL-lactate solution to the cuvette contents to give a final
sodium DL-lactate concentration of ~2.0 mM. Addition of lactate
energises the vesicles and generates a respiration dependent ΔpH (acid
inside) across the inverted vesicle membrane as H+ is pumped into the
vesicle interior. This causes a dequench of the acridine orange
fluorescence signal (see Figure 1in Representative data).

Following the establishment of a ΔpH, monitor the acridine orange
fluorescence dequench for a further ~200 sec until it stabilises.
Initiate MdtM-mediated, proton-driven antiport by adding substrate (in
this case the bile salt sodium cholate) to the inverted vesicle mixture.
We added 12.5 µl of 250 mM stock solution made up in high purity water
to give a final concentration of sodium cholate in the cuvette of ~2.0
mM. For other substrates we suggest testing a range varying from 1 mM to
100 mM to establish the concentration that gives the best dequench
signal. Upon addition of substrate, there should be an immediate
dequench (rise) of the acridine orange fluorescence emission signal (see
Figure 1a) due to dissipation of the established ΔpH as a result of
MdtM-mediated sodium cholate/H+ antiport activity and concomitant
alkalinisation of the vesicle lumen.

Record the fluorescence
dequench signal for 50 sec to allow the antiport reaction to achieve a
steady state (as observed by a plateauing of the fluorescence signal).
Addition of the protonophore CCCP to a final concentration of ~100 µM
(1.6 µl of a 100 mM stock made up in ethanol) in the assay mixture
should be performed to abolish ΔpH-driven, MdtM-mediated antiport
activity. Addition of CCCP will cause a further dequench of the
fluorescence signal as the ΔpH is dissipated (see Figure 1). Record the
fluorescence signal for a further ~50 sec then terminate the acquisition
and save the electronic data.

Decant the cuvette contents into a
suitable waste container and wash the cuvette thoroughly with ethanol
then high-purity water. Dry the cuvette carefully with compressed air.

Representative data

Figure 1. Representative measurements of the fluorescence quench/dequench of acridine orange upon addition of bile salts to inverted vesicles of E. coli TO114 cells that overproduced recombinant (a) wild type MdtM or, as a control, (b) the dysfunctional MdtM D22A mutant. Respiration-dependent generation of ΔpH (acid inside) was established by addition of sodium DL-lactate as indicated. Sodium cholate was added to vesicles as indicated to initiate the transport reaction and CCCP was used to dissipate ΔpH. The fluorescence dequench observed in the control experiment (panel b) upon addition of sodium cholate is due to antiport activity of chromosomally encoded MdtM. Fluorescence intensity is measured in counts per second (cps). Note that the fluorescence intensity you measure may differ depending upon how your instrument is set up. The traces above are representative of experiments performed in triplicate on at least two separate preparations of inverted vesicles.

Notes

To ensure reproducibility, the assays should be performed in triplicate on at least two separate preparations of inverted vesicles.

If the fluorescence signal does not quench, or enhances, the vesicles have either not maintained integrity or are not inverted and their preparation needs to be repeated.

As with all assays that rely on detection of fluorescence, robust controls must be in place to ensure that any detected transport activity can be attributed unambiguously to the protein of interest. In our experiments, we used inverted vesicles that overexpressed MdtM D22A, a dysfunctional point mutant of MdtM, as a negative control (see Figure 1b).

If the method is to be used for detection of metal ion/H+ antiport activity, the use of inverted vesicles generated from the antiporter-deficient TO114 strain of E. coli is important because at least four other transporters (NhaA, NhaB, ChaA and MdfA) present in the bacterium catalyse a monovalent metal cation/H+ exchange.

Finally, if this protocol is used for comparison of antiport activities of wild type and mutant transporters, the amount of target protein present in the inverted vesicle membranes must be quantified (usually by immunodetection methods) to ensure that any measured differences in H+ uptake are due solely to differences in the activity of the transporters and not to differences in expression levels.

Recipes

LBK agar (100 ml)
1.0 g tryptone
0.5 g yeast extract
0.745 g KCl
1.5 g agar
Make up to 100 ml with high purity water then autoclave
Add 100 μg/ml carbenicillin for selection when the solution is still liquid and warm to the touch
Add to Petri dishes under sterile conditions